This proposal is an outgrowth of our recent discovery that a tumor promoter 12-0-tetradecanoyl-phorbol-12-acetate (TPA) greatly increases the incidence of methotrexate (MTX)-resistant, colony-forming mouse 3T6 cells under conditions of a single-step MTX selection (1). Most of these resistant cells have increased dosages of the dihydrofolate reductase (DHFR) gene (1). Our specific aims in the proposed further studies are: 1) To determine to what extent other physiologically active noncytotoxic agents, such as other phorbol and non-phorbol tumor promoters and hormones, influence the incidence of MTX resistance in both mouse and human cells. 2) To study the effects of cytotoxic drugs on the incidence of cells with amplified specific genes. Both a single-step MTX selection system and an analogous system, in which cells are selected for resistance to N-phosphonacetyl-L-aspartate, a specific inhibitor of aspartate transcarbamylase, will be used in these experiments. 3) To compare absolute incidences of cells with increased dosages of the DHFR gene between difference populations of cultured cells, in particular between non-transformed and virally transformed cells. 4) To determine whether the dramatic increase of the incidence of MTX resistance in the presence of tumor promoters and certain hormones (1) can be suppressed by other substances, such as retinoic acid. 5) To develop a new assay system based on detection of free nucleoplasmic DNA copies of specific transposable elements in cultured Drosophila melanogaster cells. In this system amplification of at least some specific genes should be detectable directly thus eliminating the necessity of preliminary cytotoxic selection treatments. Results of these studies should advance our understanding of carcinogenesis and tumor promotion as related to gene amplification, provide new types of short-term assays for detection and study of carcinogenic and co-carcinogenic agents and possibly also suggest the ways to control the incidence of gene amplification-dependent drug resistance in cancer cells.